This invention relates to plasmid constructs for delivery of therapeutic nucleic acids to cells. In addition, this invention relates to methods of using those constructs as well as methods for selection of desired plasmids. Such constructs and methods are useful in human gene therapy.
Plasmids are an essential element in genetic engineering and gene therapy. Plasmids are circular DNA molecules that can be introduced or transfected into bacterial cells by transformation which replicate autonomously in the cell. They offer several advantages as vectors, the most important of which is the ability to confer antibiotic resistance to the host cell. This allows direct selection for cells that receive and maintain recombinant DNA plasmids. Plasmids also allow the amplification of cloned DNA. Some plasmids are present in 20 to 50 copies during cell growth, and after the arrest of protein synthesis, as many as 1000 copies per cell of a plasmid can be generated. (Suzuki et al., Genetic Analysis, p. 404, (1989).)
Current non-viral approaches to human gene therapy require that a potential therapeutic gene be cloned onto plasmids. Large quantities of a bacterial host harboring the plasmid may be fermented and the plasmid DNA may be purified for subsequent use. Current human clinical trials using plasmids utilize this approach. (Recombinant DNA Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6:535-548). Studies normally focus on the therapeutic gene and the elements that control its expression in the patient when designing and constructing gene therapy plasmids. Generally, therapeutic genes and regulatory elements are simply inserted into existing cloning vectors that are convenient and readily available.
Plasmid design and construction utilizes several key factors. First, plasmid replication origins determine plasmid copy number, which affects production yields. Plasmids that replicate to higher copy number can increase plasmid yield from a given volume of culture, but excessive copy number can be deleterious to the bacteria and lead to undesirable effects (Fitzwater, et al., Embo J. 7:3289-3297 (1988); Uhlin, et al., Mol. Gen. Genet. 165:167-179 (1979)). Artificially constructed plasmids are sometimes unstably maintained, leading to accumulation of plasmid-free cells and reduced production yields.
To overcome this, genes that code for antibiotic resistance phenotype are included on the plasmid antibiotics are often added to kill or inhibit plasmid-free cells. Most general purpose cloning vectors contain ampicillin resistance (.beta.-lactamase, or bla) genes. Use of ampicillin can be problematic because of the potential for residual antibiotic in the purified DNA, which can cause an allergic reaction in a treated patient. In addition, .beta.-lactam antibiotics are clinically important for disease treatment. When plasmid containing antibiotic resistant genes are used, the potential exists for the transfer of antibiotic resistance genes to a potential pathogen.
Other studies have used the neo gene which is derived from the bacterial transposon TnS. The neo gene encodes resistance to kanamycin and neomycin (Smith, Vaccine 12:1515-1519 (1994)). This gene has been used in a number of gene therapy studies, including several human clinical trials (Recombinant DNA Advisory Committee Data Management Report, December, 1994, Human Gene Therapy 6:535-548). Due to the mechanism by which resistance is imparted, residual antibiotic and transmission of the gene to potential pathogens may be of a problem than for .beta.-lactams.
Several studies have reviewed alternatives to antibiotic selection. One study made use of genes that are essential to growth of the E. coli host, such as the ssb gene, encoding the DNA single strand binding protein (Porter, et al., Bio/Technology 8:47-51 (1990)). This gene was cloned onto plasmids and used as a selectable marker in host strains lacking the chromosomal ssb gene. Because the product of the ssb gene is essential for growth, plasmid-free cells are nonviable.
A second group of studies reviewed the use of suppressor tRNA genes (Lutz, et al., Proc. Natl. Acad. Sci. USA 84:4379-4383 (1987) and Villarreal and Soo, J. Mol. Appl. Genet. 3:62-71 (1985)). One or more antibiotic resistance genes were modified to contain nonsense (stop) codons. These genes were then introduced into the host chromosome. A plasmid was then modified to contain an appropriate suppressor tRNA capable of suppressing the nonsense mutations in the antibiotic resistance genes. Introduction of the plasmid into the modified host suppresses the mutations in the resistance genes, rendering the cells able to grow in the presence of antibiotics.
Other plasmid elements which have been studied include partition elements. Such elements help stabilize plasmid maintenance independent of antibiotic selection (Hiraga, Ann Rev. Biochem. 61:283-306 (1992); Williams and Thomas, J. Gen. Microbiol. 138:1-16 (1992)). In addition, other elements promote monomerization of the plasmid. Some plasmids are prone to forming dimers, trimers and higher multimers that can reduce yield and interfere with maintenance, as well as generating a more complicated product profile. Multimer resolution elements have been employed to promote monomerization of plasmids (Eberl, et al., Mol. Microbiol. 12:131-141 (1994); Cornet, et al., J. Bacteriol. 176:3188-3195 (1994); and Summers, et al., Mol. Gen. Genet. 201: 334-338 (1985)).
In addition to elements that affect the behavior of the plasmid in the host bacteria, such as E. coli, plasmid vectors have also been shown to affect transfection and expression in eukaryotic cells. Certain plasmid sequences have been shown to reduce expression of eukaryotic genes in eukaryotic cells when carried in cis (Peterson, et al., Mol. Cell. Biol. 7:1563-1567 (1987); Yoder and Ganesan, Mol. Cell. Biol. 3:956-959 (1983); Lusky and Botchan, Nature 293:79-81 (1981); and Leite, et al., Gene 82:351-356 (1989)). Plasmid sequences also have been shown to fortuitously contain binding sites for transcriptional control proteins (Ghersa, et al., Gene 151:331-332 (1994); Tully and Cidlowski, Biochem. Biophys. Res. Comm. 144:1-10 (1987); and Kushner, et al., Mol. Endocrinol. 8:405-407 (1994)). This can cause inappropriate levels of gene expression in treated patients. In many cases, it is difficult or nearly impossible to predict when such unintended interactions will occur, unless empirical evaluation reveals the unexpected effects.